Atherosclerosis, 21 (1975) 401-408 @IIElsevier Scientific Publishing Company, Amsterdam

EFFECT

OF IRON

ATHEROSCLEROSIS

M. A. BARI* Department

AND

DEFICIENCY

ON THE

DEVELOPMENT

OF

IN CHICKS

M. ATAUR

of Biochemistry,

ANEMIA

401 - Printed in The Netherlands

RAHMAN

Jinnah Postgraduate

Medical

Centre, Karachi

(Pakistan)

(Received October 14th, 1974) (Revised, accepted January 3rd, 1975)

SUMMARY

The effect of iron deficiency anemia on the development of atherosclerosis was investigated in chicks. The control group of birds were fed a basal diet, the atherosclerosis group received 1 ‘A cholesterol, while the atherosclerosis-anemia group were given an iron-deficient diet containing 1% cholesterol. The lipid content of the aortas of the chicks in atherosclerosis and atherosclerosis-anemia groups was much greater as compared with the control group; the birds in the atherosclerosis-anemia group also developed anemia of appreciable severity in 12 weeks. Hemoglobin and PCV were taken as the index of anemia, while the lipid constituents were determined in plasma and aortic tissue for atherosclerosis. Hypercholesterolemia of almost equal severity occurred in both atherosclerosis and atherosclerosis-anemia groups. The abnormal rise of cholesterol and other lipid material in the aortas of the atherosclerosis and atherosclerosis-anemia groups of birds indicates that iron deficiency anemia did not markedly affect the development of atherosclerosis; it did not offer any protection in chickens

and, in fact, a slight potentiating

Key words:

effect was observed.

Anemia - Aortic lipid - Atherosclerosis - Hypercholesterolemia deficiency

- Iron

INTRODUCTION

Many years ago it was reported that the blood cholesterol level is low in pernicious anemia1$2. With somewhat less evidence, it has been suggested that anemia affords

* Present address: Government

Public Analyst, Multan Region, Multan, Pakistan.

402

M. A. BARI, M. ATAUR RAHMAN

protection against atherosclerosis 3. Low levels of plasma lipids, cholesterol and phospholipid were associated in most of the patients with chronic anemia due to a variety of causes4. It was suggested that this relationship is not simple and may have an important bearing on the development of atherosclerosis. Correction of anemia may not completely restore the lipid levels. The incidence of coronary disease rises sharply after the menopause and the dominant factor in the relative freedom from ischemic heart disease of premenopausal women may be their anemic state which is undoubtedly related to the negative iron balance due to menstrual losses5. Hashmi et ~1.~ have reported that the serum lipid levels of anemic male and female subjects were significantly lower than those of the control group and that these values were directly related to the severity of anemia. They further observed that improvement in the hematological status was associated with a rise in blood lipid levels. Elwood et ~1.~ noted that mild anemia had a beneficial effect on arteriosclerotic disease in women. However, Sen et ~1.8 failed to establish any correlation between hemoglobin and either plasma cholesterol or phospholipid values in anemic patients. In a comparative study, Mitchell and Mitchell9 reported that pernicious anemia does not protect against the development of atherosclerosis. In view of the variable plasma lipid levels found in anemia, the study reported here was undertaken to investigate the effect of iron deficiency anemia on the development of atherosclerosis in chicks. MATERIALS AND METHODS

One-day-old

Star

Cross

No. 566 chicks

were obtained

from

P.I.A.

Shavers

TABLE 1 COMPOSITION

OF BASAL

DIET

No.

Zngredients

%

1 2 3 4 5 6 7

Dry skim milk Soybean oil Vitamin mix& Mineral mixb Calcium carbonate Bran Corn starchc

64.0 3.0 0.1 3.1 1.8 2.0 26.0

Vitamin mix: The vitamin mix (V. Mix-Whitmoyer, UK) was purchased from P. I. A. Shavers, Karachi. When included in the diet at a level of 0.1 %, the vitamin mixture contributed all necessary vitamins required for normal growth of chicks. Mineral mix: When included in the diet at a level of 2.34x, the mineral mixture contributed the following minerals in g/kg of diet: NaCI, 10.0; MnS04. H20, 0.3; FeS04. 7Hz0, 0.65; CuSO4. 5H20,0.08; ZnO, 0.07; Co(CH&00)2.4H20,0.02; KI, 0.01; Alz(S04)3. lSHzO, 0.25; MgS04. 7Hz0, 4.0; KCI, 3.0; KaHP04, 5.0; NazMn04. 2H20, 0.01. Cholesterol was added to the basal diet as a 1.O% supplement at the expense of corn starch wherever required.

IRON DEFICIENCY

ANEMIA AND ATHEROMA

27

i

25

2

4

6 Time

6

in weeks

403

1

c 12

Fig. 1. Variation in hemoglobin and packed cell volume in control, atherosclerosis and atherosclerosis-anemia groups. Hemoglobin and PCV were determined in the blood of chicks fed with control (O), atherosclerotic (a) or atherosclerotic-iron-deficient The values are shown as mean f S.E.M.

(m) diets at different time periods.

Poultry Breeding Farm Ltd., Karachi. After 15 days, the chicks weighing between 150-175 g were selected and numbered by putting metal rings on their legs. All birds were kept under heated brooders. The temperature was maintained for 6 weeks between 95 and 100 “F, and reduced by 5°F every week thereafter. Food and tapwater were provided ad libitum throughout the study. The birds were vaccinated against Newcastle disease and Fowl pox. The chicks were fed on dry skim-milk basal diet as modified from Davis et aLlo and shown in Table 1. The birds were divided into 3 main groups: control, atherosclerosis and atherosclerosis-anemia groups. The control group were fed the basal diet with additional iron (80 mg/kg of diet). The atherosclerosis group were fed the basal diet containing 1 % cholesterol with additional iron (80 mg/kg of diet), while the third group were fed the basal diet containing 1 % cholesterol with no additional iron in the diet. The control group consisted of 48 birds, while there were 40 birds in each other group. In the control group 8 birds were killed at zero time and then 8 birds from each group were killed after 2, 4, 6, 8 and 12 weeks of feeding. Blood was collected in heparinized tubes from the overnight fasted chicks by cardiac puncture. Whole blood was used for the determination of hemoglobin and packed cell volume (PCV), while plasma was used for the determination of total and free cholesterol. After taking the blood sample, the whole aorta was removed from its root to its bifurcation. The aorta was cleaned of surrounding connective tissue and weighed.

404

M. A. BARI, M. ATAUR RAHMAN

TOTAL

FREE

Time

CHOLESTEROL

CHOLESTEROL

I” weeks

Fig. 2. Variation in plasma cholesterol levels in chicks. The mean values (III S.E.M.) of total and free cholesterol are shown in the control (0 ?? ), atherosclerosis (0 ~ 0) and atherosclerosisanemia (A---A) groups at 0, 2, 4, 6, 8 and 12 weeks of feeding normal or experimental diets.

Extraction of lipids The aortas of each group were pooled and weighed. They were cut into small pieces and homogenized in a Potter-Elvehjem homogenizer in cold Bloor’s reagent. The supernatant was used for the determination of phospholipid, cholesterol (total and free), triglycerides and free fatty acids. ANALYTICAL METHODS

Hemoglobin was determined by the cyanmethemoglobin method and packed cell volume (PCV) by the microhematocrit method. Free and total cholesterol were estimated by the method of Crawford ii, triglycerides by Stern and Shapiro’s method13 and free fatty acids by the modified method of Dole14. RESULTS

Low hemoglobin values were observed during the early life of the chicks. In the atherosclerosis-anemia group, hemoglobin concentration and PCV were decreased at 12 weeks of feeding as compared with the control group (Fig. 1). The plasma total cholesterol values for control group of birds decreased at 12 weeks (P < O.OOl), while

40.5

IRON DEFICIENCY ANEMIA AND ATHEROMA TABLE 2 VARIATION

IN LIPID

FRACTIONS

OF AORTAS

The estimations were made in the tissue homogenate prepared with Bloor’s reagent. The aortas of each group were pooled and the results expressed in mg/lOO g wet weight. Time weeks

Control group 0 2 4 6 8

12 Atherosclerosis 2 4 6 8 12

Phospholipid (total)

940 530 360 550

1550 1200 group 420 260 500 1750 1300

Atherosclerosis-anemia group 2 780 4 520 6 400 8 1750 12 1500

free cholesterol

Cholesterol

Triglycerides

830 200 230 260 460 440

370 120 150 160 370 270

460 380 340 1330 2420 4360

67.4 55.4 35.9 80.0 92.2 80.3

340 260 320

740 900

230 150 170 420 430

420 300 1140 3100 4900

59.1 73.3 55.4 96.9 158.0

900 280 340 780 980

580 120 190 460 410

590 320 950 2100 3940

88.2 36.2 55.4 110.8 138.5

Cholesterol

(Fig. 2). However, in the other groups, was very significant during this period.

increased

and total cholesterol

Free fatty

acids

(free)

the rise in both free

The effect of basal, cholesterol-supplemented and iron deficient diets on the lipid fractions in the aortic tissue showed that, in chicks receiving dietary cholesterol, aortic cholesterol levels were doubled when compared with those of the control group at 12 weeks (Table 2). A similar rise in free cholesterol was also noticed in the 2 groups. Phospholipid and triglyceride levels remained almost constant in tissue of all the groups but showed an increase with age. A greater increase in free fatty acids was found in the atherosclerosis and atherosclerosis-anemia groups. The gain in weight of the animals belonging to atherosclerosis and atherosclerosis-anemia groups was less than those of the control group. A high rate of mortality was observed in the iron deficient group, particularly between 8 to 12 weeks. The birds of this group also had depigmentation, which was more marked on their chests. The birds were comparatively inactive and showed general pallor of the skin due to iron deficiency. DISCUSSION

The anemia

produced

was that of iron deficiency

type (Fig. 1). The decrease

in

M. A. BARI, M. ATAUR

406

RAHMAN

plasma cholesterol level in the normal birds (Fig. 2) could be due to the presence of soybean oil (Table 1) in the basal diet 15. However, the cholesterol-supplemented diet caused an appreciable rise in plasma and aortic cholesterol levels at the end of 12 weeks in both

atherosclerosis

confirms

the earlier

and atherosclerosis-anemia

observations16

groups

that the frequency

(Fig.

2, Table

of lesions increases

2). This

as the con-

centration of cholesterol in the diet is raised. The esterified fraction of plasma cholesterol is increased more than free cholesterol, while the deposition in the aorta is more ester than free cholesterol (Fig. 2, Table 3). Buck and Rossiterl7 and others have also observed that with increased severity more rapidly than its ester. The increase in plasma cholesterol production

of anemia

of the disease free cholesterol was not prevented

(Fig. 2). This contrasts

is deposited

by the simultaneous

with the observations

made in human

subjects that the hypolipidemia associated with anemia may prevent or retard the development of atherosclerosis. There was no significant variation of phospholipid in the aortic tissue in all the groups but an increase with age was noted (Table 2). in Similarly, Zilversmit et d19J@ have reported that the metabolism of phospholipids the aorta is relatively independent of that in the plasma, and that in the rabbit, the aortic phospholipids are almost entirely of local synthetic origin.

REFERENCES 1 BLOOR, W. R. AND MACPHERSON,D. J., 2 MULLER, G. L., Relation of cholesterol, pernicious anemia, Anzer. J. Med. Sci., 3 MUSCHENHEIM,C., ADVOCATE, S. AND

The blood lipids in anemia, J. &of. Chem., 31 (1917) 79. lecithin phosphorous

and fatty acids to the remission of

179 (1930) 316.

HOSKINS, D. W., Inhibition of experimental hypercholesterolemia and atherogenesis by 4-amino pteroylglutamic acid (aminopterin), Circulut. Res., 8

(1960) 759. 4 RIFKIND,B. M. AND GALE, M., Hypolipidaemia in anaemia -Implication

for the epidemiology of ischaemic heart disease, Larzcer, 2 (1967) 640. 5 FRY, J., Clinical patterns and course of anaemias in general practice, &it. Med. J., 2 (1961) 1732. 6 HASHMI,J. A., AFROZE, N., BANO, S., TALAT, P., AHMAD, K., HAYAT. L. AND SYED, S. A., Pattern of blood-lipids in patients suffering from anemia, J. Pakistan Med. Ass., 19 (1969) 131. 7 ELWOOD, P. C., MAHLER, R., SWEETNAM,P., MOORE, F. AND WELSBY, E., Association between circulating haemoglobin level, serum cholesterol and blood pressure, Lancet, 1 (1970) 589. 8 SEN, S. K., MEHROTRA, R. M., KUMAR, A., CHANDRA, D., AND SINGH, M. P., Erythrocytic and plasma lipids in anaemias, Indian J. Med. Res., 56 (1968) 1232. 9 MITCHELL,1. A. AND MITCHELL,S. C., “The protective effect” of pernicious anemia on the development of atherosclerosis - A comparative study, Amer. J. Med. Sci., 247 (1964) 36. 10 DAVIS, P. N., NORRIS, L. C., AND KRATZER, F. H., Iron deficiency studies in chicks using treated isolated soybean protein diets, J. Nutr., 78 (1962) 445. 1 I CRAWFORD,N., An improved method for the determination of free and total cholesterol using the ferric chloride reaction, C/in. Chim. Actu, 3 (1958) 357. 12 OSER, B. L., In: Hawks Physiologic& Chemistry, 14th edition, McGraw-Hill, New York, N.Y.,

1965, p. 1113. 13 STERN,I. AND SHAPIRO,B., A rapid and simple method for the esterified fatty acids and for total fatty acids in blood, J. Clin. Path., 6 (1953) 158. 14 DOLE, V. P., A relationship between non-esterified fatty acids in plasma and the metabolism of glucose, J. Clin. Invest., 35 (1956) 150. 15 TENNENT,D. M., ZANETTI, M. E., SIEGEL,H., KURON, G. W. AND OTT, W. H., The influence of

IRON DEFICIENCY ANEMIA AND ATHEROMA

407

selected vegetable fats on plasma lipid concentrations and aortic atheromatosis in cholesterol fed and diethylstilb-esterol-implanted cockerels, J. Nuts., 69 (1959) 283. 16 HORLICK, L. AND KATZ, L. N., The relationship of atherosclerosis development in the chicken to the amount of cholesterol added to the diet, Amer. Heart J., 38 (1949) 336. 17 BUCK, R. C. AND ROSSITER, R. J., Lipids of normal and atherosclerotic aortas - A chemical study, Arch. Path., 51 (1951) 224. 18 BEELER,D. A., RAGLER, J. C. AND QUACKENBUSH,F. W., Effects of levels of certain dietary lipids on plasma cholesterol and atherosclerosis in chicks, J. Nutr., 78 (1962) 184. 19 ZILVERSMIT,D. B., MCCANDLESS, E. L., JORDAN, JR., P. N., HENLEY, W. S. AND ACKERMAN, R. F., The synthesis of phospholipids in human atheromatous lesions, Circulation, 23 (1961) 370. 20 ZILVERSMIT,D. B. AND MCCANDLESS, E. L., Independence of arterial phospholipid synthesis from alterations in blood lipids, J. Lipid Rex, 1 (1959) 118.

Effect of iron deficiency anemia on the development of atherosclerosis in chicks.

The effect of iron deficiency anemia on the development of atherosclerosis was investigated in chicks. The control group of birds were fed a basal die...
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